A diverse array of debilitating human neurological conditions result from dysfunction in neuron-glial interactions, as seen in diseases like Multiple Sclerosis. The intricate interactions between neurons and glia form the underlying basis of axonal ensheathment and formation of axo-glial septate junctions (AGSJs) across species. Our previous studies using Drosophila and vertebrates have established that the molecular make up of AGSJs, present in myelinated axons and fly peripheral nervous system are dependent on three key homologous proteins: CASPR [Neurexin IV (NRX IV) in flies], Contactin (CONT) and myelinating glial Neurofascin (NF155). Our more recent studies reveal that NRX IV plays an important role in axon-glial interactions and axonal ensheathment in the embryonic CNS. Here NRX IV interacts in trans with two midline glia (MLG)-specific immunoglobulin (Ig) domain containing proteins, Wrapper (WRAP) and KIRRE (a homolog of vertebrate Nephrins) to establish neuron-glial scaffolds and AGSJs. We have generated an exciting new tool that allows us to visualize the morphological and anatomical features of MLG in a way not seen before. These morphological and anatomical characteristics of MLG resemble those displayed by vertebrate oligodendrocytes. Despite their fundamental importance as signaling centers for guiding axons, surprisingly little is known about the role of MLG in ensheathment of axons and how MLG might serve as a simple, yet powerful model for understanding oligodendrocyte biology. In order to further provide impetus to the idea that Drosophila MLG and vertebrate oligodendrocytes might employ common genetic programs and molecular principles, we carried out the first forward genetic screen to identify new additional molecular players that control MLG morphogenesis and, potentially, axonal ensheathment in the CNS. We screened ~550 individual chromosomal deficiency lines across the entire Drosophila genome and identified a number of novel genes that potentially function in MLG morphogenesis and axonal ensheathment. Many of these genes have vertebrate homologs that are expressed in oligodendrocytes, but no detailed in vivo functional analysis of these genes has been carried out in any system. This proposal aims to use the powerful array of genetic, molecular and biochemical techniques available in Drosophila, together with the new genes that we have identified and tools we have generated, to address key questions related to glial biology and axonal ensheathment. The specific aims of this proposal are: (1) what are the anatomical and molecular bases of MLG morphogenesis, axon-glial interactions and axonal ensheathment in the Drosophila CNS? and (2) what are the genetic and molecular pathways that control MLG morphogenesis and how they relate to vertebrate oligodendrocyte morphogenesis? We expect that our work will provide exciting new insights into the molecular and cellular mechanisms regulating glial morphogenesis and neuron-glial interactions in vivo, and will inform us about similar mechanisms that operate in the differentiation of vertebrate myelinating glial cells. PUBLIC HEALTH RELEVANCE: Genetic and molecular mechanisms that govern glial morphogenesis, axonal ensheathment, establishment of neuro-glial scaffolds and organization of specialized electron-dense structures, the axo-glial septate junctions (AGSJs) are of utmost importance to human neuronal function. AGSJs coordinate organization of axonal domains and allow myelinated axons to propagate nerve impulses in a saltatory manner. Better understanding of the fundamental mechanisms that underlie axonal ensheathment and organization of axonal domains is critical in designing future therapeutic strategies to myelin-related diseases or demyelination disorders like for example multiple sclerosis (MS) where remyelination is required and the axonal domain structure must be preserved to allow action potential propagation and neuronal function.